Abstract |
Sox2 encodes a transcription factor that harbours a DNA binding HMG domain. It is
considered one of the key gene players for the regulation of pluripotency and early
embryonic development, and a determinant of cell fate during development and
homeostasis. During early development, Sox2 is required for the formation of the epiblast.
However, its role in stem cells is still elusive. Here, we performed a surgical ablation of Sox2
in the epiblast using a novel Conditional by Inversion Sox2 allele (Sox2COIN). The Conditional
by Inversion (COIN) method for engineering conditional alleles relies on an invertible
optimized gene trap-like element, the COIN module, for imparting conditionality. The COIN
module contains an optimized 3’ splice site-polyadenylation signal pair, but is inserted
antisense to the target gene and therefore does not alter transcription, until it is inverted by
Cre recombinase. In order to make COIN applicable to all protein-coding genes, the COIN
module has been engineered within an artificial intron, enabling insertion into an exon.
Therefore, theoretically, the COIN method should be applicable to single exon genes, and to
test this idea we engineered a COIN allele of Sox2. This single exon gene presents design
challenges, in that its proximal promoter and coding region are entirely contained within a
CpG island, and are also spanned by an overlapping transcript, Sox2Ot, which contains
mmu-miR1897. Here, we show that despite disruption of the CpG island by the COIN
module intron, the COIN allele of Sox2 (Sox2COIN) is phenotypically wild type, and also does
not interfere with expression of Sox2Ot and miR1897. Furthermore, the inverted COIN allele
of Sox2, Sox2INV is functionally null, as homozygotes recapitulate the phenotype of
Sox2ßgeo/ßgeo mice, a well-characterized Sox2 null. Lastly, the benefit of the eGFP marker
embedded in the COIN allele is demonstrated as it mirrors the expression pattern of Sox2.
Epiblast-inversion of the Sox2COIN allele generates normal heterozygote Sox2INV/+ adult
animals and Sox2INV/+ (H) haploinsufficient mutant embryos. Sox2INV/+ (H) haploinsufficient and
Sox2INV/mosaic embryos exhibit heart-looping defects, suffer from bradycardia, myocardium
malformation and bleeding and die around E11. Sox2INV/+(H) haploinsufficient and
Sox2INV/mosaic embryos exhibit hydrocephaly, exacerbated and aberrant migration of neural
crest cells in the branchial arches and the frontonasal region, with no effect on cranial nerve
formation but abnormal frontonasal development. We propose that Sox2 acts as a rheostat
of the epithelial to mesenchymal transition during neural crest development.
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